Project Summary/Abstract: The goal of the project proposed in this application is to replace retinal neurons that are lost to disease or injury with new neurons by stimulating the resident glial cells of the retina, the Mller glia (MG), to undergo neurogenesis. Diseases ranging from glaucoma, macular degeneration, and Retinitis Pigmentosa, destroy various neuronal populations in the retina and result in visual impairment or blindness for millions of people around the world. Sight is arguably the most important sense humans possess, and there are currently only very limited clinical options to restore vision once the cells in the retina have degenerated. Amazingly, many non-mammalian species have the ability to regenerate their retinal neurons from MG and restore visual function after damage. Recent discoveries elucidating the mechanisms by which these species repair their retinas have led to my proposed project to reproduce MG-mediated retinal regeneration in mice. Generating immature retinal neurons from MG has recently been achieved in young mice using transgenic MG directed expression of the proneural transcription factor Ascl1. In my preliminary studies, I have found that the combination of MG-specific Ascl1 expression and the addition of a histone deacetylase inhibitor enables MG to generate new neurons in a damaged adult mouse retina. This exciting finding now opens the way for many additional studies to investigate the long-term viability and functionality of the regenerated neurons. The following three proposed Aims will 1) determine whether the MG-derived neurons are stable for longer periods of time, 2) determine if they retain some of their glial characteristics, and 3) determine if there are methods for more effectively maturing these MG-derived neurons. Using state-of-the-art imaging techniques, such as super-resolution Zeiss Airyscan confocal microscopy, and serial block-face scanning electron microscopy I will address the extent to which the MG-derived neurons achieve mature neuronal morphology and connectivity. Using whole-cell electrophysiology and measuring the MG-derived neuron's response to light will address neuronal functionality and maturity. Lastly, using single-cell mRNA-sequencing on FACS-purified MG-derived neurons will address the extent to which the MG-derived neurons develop a pattern of gene expression similar to known mature retinal cell neuronal types, or whether they retain some degree of glial gene expression. By utilizing these techniques to characterize the neuronal state of the cells, the experimental paradigm can then be modified, as described in the Research Strategy, to determine if MG-derived neurons can be driven to a more mature state. The proposed study highlights and expands upon the only known protocol for generating new neurons in the adult mammalian retina, and presents a unique opportunity to expand the field of regenerative medicine and push it closer to a potential clinical therapy.